There have been proposals to develop new sequencing technologies based on single-molecule measurements. For example, sequencing strategies have been proposed that are based upon observing the interaction of particular proteins with DNA or by using ultra high resolution scanned probe microscopy. See, e.g., Rigler, et al., J. Biotechnol., 86(3):161 (2001); Goodwin, P. M., et al., Nucleosides & Nucleotides, 16(5-6):543-550 (1997); Howorka, S., et al., Nature Biotechnol., 19(7):636-639 (2001); Meller, A., et al., Proc. Nat'l. Acad. Sci., 97(3):1079-1084 (2000); Driscoll, R. J., et al., Nature, 346(6281):294-296 (1990).
Recently, a sequencing-by-synthesis methodology has been proposed that resulted in sequence determination, but not with consecutive base incorporation. See, Braslavsky, et al., Proc. Nat'l Acad. Sci., 100: 3960-3964 (2003). An impediment to base-over-base sequencing has been the use of bulky fluorophores that can sterically hinder sequential base incorporation. Even when the label is cleaved, some fluorescently-labeled nucleotides sterically hinder subsequent base incorporation due to the residue of the linker left behind after cleavage.
A need therefore exists for nucleotide analogs having reduced steric hindrance, thereby allowing the polymerase to produce greater read-length from each template.